According to current estimates from the American Diabetes Association, there were approximately 18.2 million diabetics in the U.S. in 2002 (6.3% of the population), with 910,000 to 1,800,000 afflicted with Type I insulin-dependent diabetes. In an envisioned alternative therapeutic approach, adult cells are retrieved by a biopsy from the patient and engineered ex vivo to acquire beta cell characteristics. The cells are then re-implanted in the patient, either without or, preferably, after incorporation in a 3-D substitute for localization, retrievability, and improved maintenance of cell function. It would be most desirable if some of the engineered cells were preserved for later use by the same individual, as needed. A significant hurdle for deployment of such constructs is an effective means for long-term storage and transportation to permit worldwide product distribution. Ice formation within tissue matrices causes matrix damage in three- dimensional structures during "traditional" cryopreservation by freezing. Preliminary studies demonstrated that vitrification is a feasible storage method for tissue-engineered pancreatic substitutes. Effective cryopreservation of cells and tissues requires that they retain their genetic and phenotypic characteristics after thawing. For example, a successful cryopreservation protocol would allow genetically modified cells, prior to preservation, to express the same proteins and at the same levels after thawing. In this proposal, testing of vitrification as a storage method for tissue-engineered pancreas is extended to include genetically engineered pancreatic models (human HepG2 cells). The effects of cryoprotective agent cytotoxicity on gene expression, insulin-secretion, and cell viability will be evaluated using HepG2 cell suspensions. In addition, several new methods of vitrification using molecular ice control will be screened using 3-D constructs and the most effective formulations will be tested in a different gene transduction 3-D model of non-(3 cells (human primary recombinant hepatocytes) to test the efficacy of the optimized cryopreservation method. Finally, a small pilot animal study will test the efficacy of the cryopreservation protocols in vivo. The overall goal of this proposal is to characterize the effect of cryopreservation on the recombinant gene expression, insulin secretion and cell viability from recombinant cells that can be incorporated in pancreatic substitutes design. [unreadable] [unreadable] [unreadable]